Micro-Morphology of Achillea Clypeolata

Journal of Applied Pharmaceutical Science 02 (08); 2012: 165-170

ISSN: 2231-3354 Micro-morphology of Achillea clypeolata: Received on: 12-07-2012 Revised on: 12-08-2012 Accepted on: 17-08-2011 contribution to the pharmacognostical profile DOI: 10.7324/JAPS.2012.2828

Anely Nedelcheva

ABSTRACT

Achillea clypeolata Sibth. & Sm. is Balkan endemic species, commonly known as

yellow yarrow and traditionally used in Bulgarian folk medicine. The purpose of this study was to establish pharmacognostical profile of A. clypeolata, including macroscopic and microscopic morphological characteristics. The study also aims to propose a method for micromorphological

investigation and pharmacognostical identification. As a result were established morphological

characters necessary for identification of Achilleae clypeolatae flos and Achilleae clypeolatae

herba – morphology of stem leaves (lanceolate in shape, pinnately lobed-margin, acute-apex Anely Nedelcheva Department of Botany with lamina), ligule petals (2.7-3 mm), involucre bracts (4.5x2 mm) and achenes (1.5-1.7 mm);

Sofia University “St. Kliment micromorphology of lamina (110-130 µm), epidermal cells, stomata, glandular, non-glandular Ohridski”, Faculty of Biology trichomes and 3-colporate, echinate pollen grains (20.5-20.8 µm). This study presents new blvd. Dragan Tzankov 8, Sofia–1164 micromorphological data. The used staining method dyed various tissues; makes visible the Bulgaria outlines of structures, tissues and some inclusions; allows by one procedure to visualize

important diagnostic features; unlike many other methods, allowing pollen to be used as a characteristic of herbal substances. The results are useful for quality control methods for

medicinal plant materials and contributes to the implementation of good practices for plant

identification for the herbal industry, food, nutrient supplements and other plant based products.

Keywords: Yellow yarrow, Herba, Flos, Pharmacognosy, Balkan endemic, Bulgaria.

INTRODUCTION

Genus Achillea L. is one of the most polymorphic genera and difficult for interpretation

in the family Asteraceae and is object of recent taxonomical comments on the basis of

multidisciplinary studies (Ehrendorfer and Guo, 2005). Biosystematic studies of Achillea have a tradition of long standing in Bulgarian botany (Kuzmanov, 1984, Nedelcheva, 1998, Saukel et al.,

2003). According to the latest data, the genus comprises about 20 (3+17) species of perennial

herbs distributed in Bulgaria, belonging to two sections: sect. Anthemoideae (DC.) Heimerl s.l. and

sect. Achillea s.l. [= sect. Millefolium (Adanson) Koch s.l., incl. Sect. Filipendulinae (DC.) Afan.]. For Correspondence In Bulgaria are known 6 species with yellow flowers, (A. clypeolata Sibth. & Sm., A. coarctata Anely Nedelcheva Poir., A. pseudopectinata Janka s.lat., A. chrysocoma Friv., A. thracica Velen. and A. ochroleuca Phone: (+359 2) 8167 /304/ Ehrh.) (Saukel et al., 2003, Nedelcheva and Tzonev, 2006). Email: [email protected]

Journal of Applied Pharmaceutical Science 02 (08); 2012: 165-170

Achillea clypeolata is a Balkan endemic species and MATERIALS AND METHODS ranges from North and Central Greece to South Albania, Preparation of the plant material and samples Macedonia, Serbia, Bulgaria, South-east Romania, West and Aboveground parts of the plant are used herbal substance: European Turkey. Together with Achillea aegyptiaca L. (incl. Achilleae clypeolatae flos and Achilleae clypeolatae herba Achillea taygetea Boiss.) they form the dominantly diploid and (Nikolov, 2006). The present study is examination of the dry and Balkan centered group of A. clypeolata, characterized by grounded plant material collected from natural areas and morphometric and phytochemical features. A. clypeolata is most commercial herb samples (product contains dried herbs) (Table 1). morphological close to Achillea coarctata (Richardson, 1976, Medium representative sampling were separated and processed. Saukel et al., 2003). Warm water (up to 30ºC) is poured over a certain amount of dried Among species with yellow flowers, mostly A. clypeolata herb, covered, and allowed to steep for 20-40 minutes. For commonly known as “yellow yarrow” and traditionally used in temporary or permanent microscope observation small amount of Bulgarian folk medicine to treat hemorrhoids, wounds, bleeding, wet herbal fragments were placed on microscope slides and mixed gastro-intestinal atony, bed wetting, kidney inflammation, with 1-2 drops from differential staining mixture and fixed with amenorrhoea and inflamed gums, liver deseases. Its medicinal cover slips. The cover-slips of slides intended for storage were effect is like white yarrow, which is more widespread and better sealed with paraffin wax to prevent excessive evaporation of the known in folk medicine. Bulgarian folk names: “zhult ravnets”, mounting medium. The mounts were kept in laboratory oven at “strumski ravnets”, “div pelin”, “zhulta eniovitsa”, “skalen 50ºC for 24 hr. Mounts stored for over a 6 months (refrigerator 2- ravnets”, “ramchets”, “glavoch” and “gushterka”. The large 6ºC) showed no loss of staining. number and variety of folk names showed that the plant is well known and wide used. They are based mainly on its yellow color, Table 1: List of the specimens used in morphological investigations.

shape of the inflorescence, bitter taste like mugwort and rocky Natural areas Commercial herbal samples habitats (Ahtarov et al., 1939; Nedelcheva and Dogan, 2009). herbal samples Some of the current research focuses on the AN/1/645; AN/1/304; Bilki Eood/ 2010 pharmacological properties of the yellow yarrow (Konyalioglu and Achilleae clypeolatae AN/1/077; Karmen/2005 flos Karamenderes, 2005; Karaalp et al., 2009). Significantly more are AN/1/532; Suni Yambol EOOD/2010 the data about white yarrow as herbal drug (herbal substance) AN/1/062 (Rauchensteiner et al., 2004; Nirmala and Karthiyayini, 2010). AN/2/005; AN/2/112; Of central importance in the practice is correct Achilleae clypeolatae Bilki Eood/ 2010; Karmen AN/2/452; herba Solara /2009 identification of the species concerned, whether in the fresh, dried AN/2/33; or powdered state, because one of the criticisms of herbal medicine AN/2/076 is lack of standardization and quality control profiles. The macroscopic and microscopic description of a medicinal plant is The stain was prepared according to Alexander (1969) the first step towards establishing the identity and the degree of from various constituents: malachite green, glycerol, phenol, purity of such materials (identity tests) and should be carried out chloral hydrate, acid fuchsin, orange G, glacial acetic acid, before any tests to be undertaken (WHO, 1998). Therefore, more glycerol, 95% alcohol, distilled water. The amount of glacial acetic nowadays studies are conducted on micromorhology of herbal acid added to mixture is (4 ml to 100 ml stain mixture), because drugs for their correct identification (Jayeola, 2009; HMPC, 2010; thick-walled and spiny pollen grains as well as 5 mg acid fuchsin HMPC, 2010a). For A. clypeolata there is no pharmacopoeial additionally with aim to improve contrast (Nedelcheva, 1998). In monograph and in control of starting herbal material (including the staining process malachite green is used for staining cell walls mother plant identification) should be followed detailed and pollen walls. specification of the quality controls (EDQM, 2011; Nikam et al., Acid fuchsin in the present mixture is included for 2012). The wide use of the yellow yarrow as a herb in Bulgaria, its staining the protoplasm and also colored non aborted pollen grains place in folk medicine and its use today in the herbal remedies, in red to red-purple. These characteristics gave reason to along with limited data as a plant substance justify the purpose of experiment with this stain solution and on plant tissues, not only to this study: to establish pharmacognostical profile of A. clypeolata, differential staining of aborted and non aborted pollen grains which including macroscopic and microscopic morphological was the target of Alexander (1969). characteristics. The study also aims to propose a method for For morphological investigations related size measures, micromorphological investigation and pharmacognostical twenty plant individuals from each population or samples were identification of the herbal substances which is informative, fast studied. Dry herbal substances were analyzed and photographed and with easy laboratory performance as well as applicable in the using the light microscope Olympus BX with digital camera system of herbal gathering and herb production, preparation and Olympus SP-510UZ. implementation of marketing, quality control of herbal Voucher specimens was deposited in the personal author preparations, food, nutrient supplements and other plant based collection in Department of Botany and in the Herbarium of products. University of Sofia (SO).

Journal of Applied Pharmaceutical Science 02 (08); 2012: 165-170

Study area Based on this preliminary analysis was performed Bulgaria (42°41′0″N, 23°19′0″E) is a country in the micromorphological investigation on most involved characteristics Balkans in south-eastern Europe. It borders five other countries: with diagnostic value and last but not least susceptible to dying by Romana to the north (mostly along the River Danube), Serbia and the proposed method. Inflorescences are yellow, dense, flat-topped the Republic of Macedonia to the west, and Greece and Turkey to and 3-5(7) сm wide corymbs. They are composed of many 50- the south. The Black Sea defines the extent of the country to the 150(250) flower heads (capitulum) obovate in outline and variable east. Phytogeographically, Bulgaria straddles the Illyrian and in range 3-4(6) mm long and 2-3(4) mm wide. Capitulim is Euxinian provinces of the Circumboreal region. composed of both ray (2-5) and disk (10-20) florets; in the base Bulgarian flora comprises 159 families, 906 genera and with conic or convex receptacle, surrounded by imbricate bracts in 4030 species, 12.8% are endemics (Petrova and Vladimirov, 2010). several series (Fig. 2). There are about 770 species of medicinal plants constituting 20% of the Bulgarian flora. Of these, 200 are currently in use and over 250 herbal drugs are derived from them and presently used. The plant is included in the list of medicinal plants in Bulgaria (MOEW, 2000). Achilleae clypeolatae flos is wild collected herbal substance (drug), wich export quantities officially declared by licenses (2001-2005) is range 100-250 kg. Achilleae millefolii flos is more than 36 000 kg (Evstatieva et al., 2007). Both species are not cultivated in Bulgaria.

RESULTS AND DISCUSSION

The structure of the herbal substances was established Fig. 2: The morphology and structure of capitulum. Bar = 1mm. after weight examination of the plant fragments with different Involucrum is obovate-oblong, imbricate, 2-3(4) mm long origin. In both of them (flos and herba) is counted prevalence of and 2-3(4) mm wide, squamous, with narrow transparent the involucrum and florets fragments, followed by leaves membranous margin or without such, yellowish to brownish, outer fragments and with well distinguished glandular and non glandular bracts are shorter and narrower than outline cover bracts (Fig. 3). trichomes as well as pollen grains (Fig. 1). In the involucrum bracts are visible elongated epidermall cells, on

the abaxial surface with ± covering and glandule trichomes similar to those on leaves. Margins of the involucrum bracts are irregular as outline (Fig. 4).

Fig. 3: The morphology of involucrum bracts. Bar = 1mm.

Fig. 1: Structure of the composition of the herbal substances Achilleae clypeolatae flos (A) and Achilleae clypeolatae herba (B). Fig. 4: Fragment of lamina with non-glandular trichomes. Bar = 0.25mm. Journal of Applied Pharmaceutical Science 02 (08); 2012: 165-170

Ray florets are 2-4(5) per head, pistillate, fertile; corollas Rauchensteiner et al., 2004). In this study, the herbal samples were are yellow, lamina is elliptical 0.5-1.0(1.5) x 1.5-2(3) mm. Ligule tested for proazulene positive glands and gave the negative result. is deeply tripartite in outline, long to ¼ of the length of The missing colour indicates a different composition within the involucrum. Ray florets are morphologically variable (Fig. 5 ). On sesquiterpene lactones. These data confirm previous phytochemical the outside of the corolla tube are visible glandular trichomes reports for sesquiterpene content and there is no info for similar to those on leaves. Adaxial epidermal cells of the ligulae proasulenes (Ivancheva and Nedelcheva, 1998, Nedelcheva and are isodiametric, papillose and striated and with elongated abaxial Ivancheva, 1998, Todorova and Tzankova, 1999, Saukel et al., epidermal cells; Stigma is 2-lobed, with papillosed surface. Forked 2003, Nikolova and Ivancheva, 2006). stigma lobes are 175-225 µm long (Fig. 6). The yellow color of ray florets is a good diagnostic feature, but in some habitats, in some hybrids can be pale yellow as well as in improperly stored herbal substance (Nedelcheva , 1998).

Fig. 7: Micromorphology of undeveloped achena. Bar = 1mm.

Fig. 5: The morphology of ray florets. Bar = 1mm.

Fig. 8: Glandular trichome (s) in ray florets outline without color. Bar = 50µm

Fig. 6: Stigma (two-lobed) with papillosed surface. Bar = 0.1mm.

Achenes in the herbal substances are not maturated. They are light brownish in color, not well developed white margin and their average length/width ratio is 2. Undeveloped achena is 0.9- 1.1 mm long and 0.5 mm wide, obovoid-oblong in shape, translucent, with a ring of sclereids at apex and base, without glandular trichomes (Fig. 7 ). Taxonomic value of the achene Fig. 9: Glandular trichome (s) in ray florets outline with blue-green color. Bar = micromorphology and anathomy is well known for Achillea 50µm species (Akcin and Akcin, 2010). Glandular trichomes are biseriate and have at full From disc florets are well distinguished elongated growth a subcuticular large spherical chamber a space coming out epidermal cells and mesophyll with small oxalate druses (Fig.10). when the two layers of the cuticle become detached from the In outline have papillose triangular lobes of corolla. Biseriate pecto-cellulosic wall (Fahn, 2000) (Fig. 8). Some of the glandular glandular trichomes are frequent on the outside of the floral tube trichomes appear in blue (Fig. 9), blue-green or brownish, whereas and rarely on the lobes. Connate anthers contain pollen into the any other or missing colour. Proazulenes can be easely detected by interior of the anther tube. Anther sac is filled with fertile pollen simply heating some florets in a mixture of chloralhydrate (60%) grains. In Fig. 11 are visible pollenkitt fragments (oily grains in and phosphoric acid (85%). During this process proazulenes are yellow) in which the pollen grains are suspended during transformed to coloured chamazulenes (Saukel, 1993, the period from anther opening (Pacini and Hesse, 2005). Journal of Applied Pharmaceutical Science 02 (08); 2012: 165-170

the composition of the both herbal substances: flos and herba. The stem leaves are up to 4-7 on the flowering stem, oblong to lanceolate in shape, pinnately lobed to pectinate, 3-5(10) cm in length and 1-1.5(2) cm in width, grey-green in color. When hang drying the aerial parts the stem leaves are often scattered and found in very small quantities in the samples. In contrast is Achilleae millefolii herba, where the leaves are significant part in the final drug, and therefore the leaves are subject to more detailed studies (Nirmala and Karthiyayini, 2010). Non-glandular trichomes are also frequently occurring in the epidermal cells of the leaves and stem. The trichomes are uniseriate, of varying length, Fig. 10: Micromorphology of disc florets. Bar = 50µm. commonly with swollen basal cells or seated on pedestals of several epidermal cells (Fig. 13). It is 650 μm in length. Swollen basal cell is 30 μm in diameter and tip of the trichome is 15 μm in diameter. Epidermal cells are lobed and amoeboid in outline. The stoma is consisting of an elliptical pore in the epidermis and surrounded by two specialized, kidney-shaped epidermal cells (Fig. 14). These data confirm previous reports for Achillea trichomes morphology (Nedelcheva, 1998, Nirmala and Karthiyayini, 2010).

Fig. 11: Stained pollen grains in nondehiscent anthers with an oily, sticky materal (pollenkitt). Bar = 50µm

After applied staining fertille and sterille pollen grains are well distinguished. The aborted pollen is stained blue (blue- green) and the non aborted pollen grain is red to red-purple, because of protoplasm (Fig. 12). In both cases the pollen wall is well visible. Pollen grains are tricolporate sheroidal with spiny exine, and 18-22 µm in diameter (not include spines). According to Fig. 13: Non-glandular trichomes on the surface view. Bar = 50µm Nedelcheva (1998), this pollen size meets the measured size of pollen prepared by acetolysis method of Erdtman (1960). Presented results showed the significance of the pollen morphology in herbal substance identification.

Fig. 14: Fragment of abaxial epidermis with stomata. Bar = 50µm

CONCLUSION

Determination of the diagnostic characters (macro- and

Fig. 12: Aborted and nonaborted pollen grains. Bar = 50µm. micro morphological) is the referential information for the correct identification of the herbal substances. This study presents new In the studied samples participation of leaves is limited micromorphological data about Balkan endemic A. clypeolata and which is according with general morphology of A. clypeolata. The is contribution to pharmacognostical profile of yellow yarrow basal leaves give the main biomass of leaves, but they are not in based herbal substances. The used staining method does not require Journal of Applied Pharmaceutical Science 02 (08); 2012: 165-170

complicated preparation of herbal samples for testing. By contrast Ivancheva S. and Nedelcheva A. (1998) Exudate flavonoid aglycones of Achillea species from sect. Millefolium and sect. it is dyed various tissues; makes visible the outlines of structures, . Filipendulinae - Comparative study. In N. Randelovic (Ed.) Zbornik tissues and some inclusions; allows by one procedure to visualize Radova V Symposium o Flori Jugoistočne Srbije (pp. 183-189). Niš: important diagnostic features; unlike many other methods, Pergament allowing pollen to be used as a characteristic of herbal substances. Jayeola A.A. Micromorphological study of plant fragments in The present paper describes a new utilization of a staining some powdered medicinal plants. J Med Plants Res. 2009; 3(5): 438-442 Karaalp C., Yurtman A.N. and Karabay Yavasoglu N.U. technique originated by Alexander (1969) and modified for this Evaluation of antimicrobial properties of Achillea L. flower head extracts. study. 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